Characterization of acute myeloid leukemia with MLL ... - Nature

Leukemia (2000) 14, 1025–1030  2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00 www.nature.com/leu

Characterization of acute myeloid leukemia with MLL rearrangements – no increase in the incidence of coexpression of lymphoid-associated antigens on leukemic blasts H-F Tien1, C-H Hsiao1, J-L Tang1, W Tsay1, C-H Hu1, Y-Y Kuo1, C-H Wang1,2, Y-C Chen1,2, M-C Shen1,2, D-T Lin2,3, K-H Lin3 and K-S Lin3 Departments of 1Internal Medicine, 2Laboratory Medicine, and 3Pediatrics, National Taiwan University Hospital, Taipei, Taiwan

MLL gene rearrangements are associated with coexpression of myeloid- and lymphoid-associated antigens on leukemic blasts and a dismal outcome in acute lymphoblastic leukemia (ALL). Whether the same conditions can apply to acute myeloid leukemia (AML) is not quite clear. Rearrangements of the MLL gene were analyzed on 113 patients with newly diagnosed de novo AML in a single institution. Sixteen (14%) of them showed rearranged bands by Southern blot analysis, including three (50%) of six infants, three (14%) of 21 children between 1 and 15 years and 10 (12%) of 86 adults. MLL rearrangements were not only detected in M5 (four of 12 patients, 33%) and M4 (six of 31, 19%) subtypes but also in other non-M4–M5 AML (six of 70, 9%), including M1, M2 and M7, but not M3 subtype. Seven patients had chromosomal abnormalities involving 11q23, but nine did not. The latter comprised three (6%) of 48 patients with normal karyotype, one with t(8;21), none with t(15;17), inv(16) or t(9;22), and four (15%) of 27 with cytogenetic aberrations other than those specific structural abnormalities. In contrast to ALL, AML patients with MLL rearrangements did not tend to coexpress lymphoid- and myeloid-associated antigens simultaneously on leukemic blasts and have similar outcome as those without the gene rearrangements. Leukemia (2000) 14, 1025–1030. Keywords: MLL; acute myeloid leukemia; lymphoid antigen

Introduction Nonrandom chromosomal abnormalities are nowadays one of the most significant prognostic factors in acute leukemia and are applied increasingly to risk-adapted therapeutic stratification.1,2 Structural abnormalities involving chromosome band 11q23 are among the most common cytogenetic aberrations in acute leukemia.3,4 Recently, a gene named MLL (for mixed lineage leukemia or myeloid-lymphoid leukemia) that spans the 11q23 breakpoints has been cloned which is strongly homologous to the Drosphila trithorax gene and presumably coding for a transcription factor.5–8 MLL gene is rearranged in leukemic cells from most patients with various 11q23 translocation, which involves at least 50 partner genes at different chromosome loci,9–12 but is usually not affected in 11q23 deletions.13,14 The gene rearrangements can also be demonstrated in some acute leukemia without cytogenetic abnormalities at 11q23.15,16 The study of MLL gene rearrangements can more specifically and sensitively define a subgroup of acute leukemia than conventional cytogenetic analysis of 11q23 abnormalities. In acute myeloid leukemia (AML), MLL rearrangements are most frequently disclosed in French–American–British (FAB) M4 and M5 subtypes with an overall incidence of 10% to 30%.15,17 Little is known about their frequency in other subtypes of AML. The presence of MLL rearrangements were Correspondence: H-F Tien, Department of Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, Taiwan, ROC; Fax: 886 2 23959583 Received 12 July 1999; accepted 4 February 2000

found to be associated with coexpression of myeloid antigen on lymphoblasts and correlated with poor prognosis, regardless of presenting age, in acute lymphoblastic leukemia (ALL).18,19 Fewer studies have been conducted to characterize the immunophenotype of AML with MLL rearrangements. The prognostic implication of the gene rearrangements in AML also remains to be clarified. AML patients with balanced 11q23 breakpoints and/or MLL rearrangements were found to have shorter disease-free survival than control patients in one study,20 but the gene rearrangements showed no prognostic significance in M4 or M5 subtype of AML in another report.15 In this study, rearrangements of MLL gene were analyzed on 113 newly diagnosed primary AML and were correlated with the clinical manifestations, biological characteristics and outcome of the disease. Materials and methods

Patients MLL gene rearrangements of the leukemic cells were studied at presentation on 113 newly diagnosed patients with primary AML in National Taiwan University Hospital. No patients had a history of prior treatment with chemotherapy or radiotherapy, and none had a history of other hematologic disorders. The median age was 35 years, ranging from 4 months to 82 years. There were 54 males and 59 females. The diagnosis and classification of AML were based on the criteria established by the French–American–British (FAB) Cooperative Study Group.21,22

Cytogenetic study Chromosome analysis was performed as described previously.23 Briefly, bone marrow (BM) cells were harvested either directly or after 1–3 days of unstimulated culture. Metaphase chromosomes were banded by the conventional trypsin-Giemsa banding technique and karyotyped according to ISCN.24

Immunophenotyping A panel of monoclonal antibodies to myeloid-associated antigens including CD13, CD33, CD14, CD11b, CD15, CD41 and glycophorin A, as well as to lymphoid-associated antigens including CD2, CD5, CD7, CD10, CD19, and CD20, and to lineage-nonspecific antigens HLA-DR and CD34 were selected to characterize immunologic phenotypes of leukemic cells. Expression of surface antigens on leukemic cells was shown by an indirect immunoalkaline phosphatase method.25 Samples in which more than 20% of leukemic cells were posi-

MLL-rearranged AML H-F Tien et al

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tively stained with the antibody were considered positive for that marker.

Detection of MLL gene rearrangements Mononuclear cells isolated from bone marrow aspirates were cryopreserved on the same day of sample collection with a few exceptions. MLL gene rearrangements were detected by Southern blot analysis.26 Genomic DNAs were digested with restriction enzymes BamHI and HindIII in conditions recommended by the supplier (Boehringer, Mannheim, Germany). They were size-fractionated by electrophoresis, transferred to Hybond membrane (Amersham International, Amersham, UK) and then hybridized with 32P-dCTP-labelled probe. The probe used was a 740-bp cDNA fragment of MLL extending the breakpoint cluster regions between exons 5 and 11 (kindly provided by JR Downing, St Jude Children’s Research Hospital, Memphis, TN, USA).

Detection of immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements Analysis of rearrangements of Ig heavy-chain and TCR ␤-chain genes was performed as described previously.26 Five micrograms of each DNA were digested with restriction enzymes BamHI and EcoRI. The probes used were DNA fragments from a TCR ␤-chain gene (BglII-digested C␤2 region of TCR␤ gene, kindly provided by Dr TW Mak, Amgen Institute, Toronto, Ontario, Canada); and Ig gene JH fragment (BamHI– HindIII fragment of JH region, kindly provided by Dr P Leder, Harvard Medical School, Boston, MA, USA).

Statistical methods Continuous variables were compared by Wilcoxon rank-sum test and discrete variables were compared by ␹2 or Fisher’s exact test. Curves of survival and duration of complete remission were plotted by the Kaplan–Meier method; differences between curves were analyzed by the log-rank test. The implications of the presence of MLL rearrangements on complete remission duration and overall survival in AML patients were evaluated using Cox regression model. Statistical analyses were performed using the SPSS for Windows Release 7.0 (SPSS Inc, Chicago, IL, USA).

Figure 1 (a) A representative film of Southern blot analysis on BamHI-digested DNA from AML patients showing MLL rearrangements on lanes 1 and 4. (b) Another film of Southern blot analysis on HindIII-digested DNA showing MLL rearrangements on lanes 1 and 2. (c) Southern blot analysis on one patient with t(8;21) showing germline configuration of MLL at diagnosis (lane 2), but a rearranged band at relapse (lane 1). C, normal control.

were no significant differences in other clinical manifestations between the two groups of patients. Among the 113 AML, there were 24 M1, 33 M2, 9 M3, 31 M4, 12 M5, 1 M6 and 3 M7 subtypes. Patients with M5 subtype of AML had the most frequent MLL gene rearrangements (four cases, 33%), followed by M4 (six, 19%), M2 (three, 9%) and M1 (two, 8%). One M7 patient also had MLL gene rearrangements. None of M3 and M6 subtypes of AML showed the gene rearrangements. Among the 16 patients with MLL gene rearrangements, 10 (63%) had monocytic leukemia M4 or M5 subtypes, compared with 33 (34%) of the 97 patients without the gene rearrangements (P = 0.03). Other hematologic data were similar between the two groups of patients.

Results

Leukemia

Correlation of MLL rearrangements with clinical and hematologic features

Correlation of MLL rearrangements with chromosomal changes

A total of 16 patients (14%) was found to have MLL rearrangements in the leukemic cells (Figure 1). Pseudo-rearrangement of MLL gene caused by cell apoptosis27–29 was carefully excluded. The clinical and hematologic features of the patients with MLL rearrangements were compared with those without the change (Table 1). Among the 113 patients who had the gene analysis, 86 were adults (⬎15 years), 21 were children between 1 and 15 years and six were infants. Infants had the highest incidence of MLL rearrangements (three cases, 50%), compared with that of children between 1 and 15 years (three cases, 14%) and that of adults (10 cases, 12%). There

Cytogenetic study was performed on leukemic cells from 108 patients. All but one had adequate metaphase cells for analysis. Fifty-nine patients (55%) showed clonal chromosomal abnormalities. Rearrangements involving chromosome 11q23 were detected in nine patients (Table 2); three had t(9;11), one t(11;17), two t(6;11) and three, deletion. Seven (78%) of them had MLL gene rearrangements, including all six with 11q23 translocation and one (33%) with deletion. All 23 patients with other specific structural aberrations, including t(15;17), inv(16), t(8;21) and t(9;22), showed germline configuration of the MLL gene, with the exception of one patient who had


Table 1

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Correlation of MLL gene rearrangements with clinical and hematologic features

MLL rearranged No. of cases Median age (year) Age group (No. of cases) Infant Children ⭓1 yr, ⭐15 yr Adults Hepatomegaly (%) Splenomegaly (%) Lymphadenopathy (%) CNS involvement (%) FAB M4–M5 subtype (%) Hb (median, g/dl) WBC (median,/␮l) Platelet (median, ×103/␮l) Lactate dehydrogenase (U/l) CR rate (%) CR duration (median, months) Survival (median, months)

16 22 3 3 10 38 32 25 0 63 7 20 580 56 1074 50 6 7

Table 2 Correlation of MLL gene rearrangements and chromosome change

Cytogenetics subtype 11q23 del(11)(q23) t(11)(q23) Normal t(8;21) t(15;17) inv(16) t(9;22) Others Not done Total

Total No. of patients

No. with MLL rearrangement

Positive rate

9 3 6 48 8 7 6 2 27 6b 113

7 1 6 3 1 0 0 0 4a 1 16

78% 33% 100% 6% 13% 0 0 0 15% 17% 14%

a

del(11)(p11.2), del(20)(q11.2), t(1;22)(p13;q13), t(1;19)(p34; p13). Including the one without metaphase.

b

t(8;21). MLL gene rearrangements were detected in four (15%) of the 27 patients with chromosomal abnormalities other than those specific ones mentioned above (Table 2). Three (6%) of the 48 patients with normal karyotype also showed the same gene abnormality.

Correlation of MLL rearrangements with immunophenotypes and rearrangements of Ig and TCR genes Immunophenotyping was performed on leukemic cells from 110 patients. There was no significant difference in the incidence of surface antigen expression of leukemic cells between AML patients with MLL rearrangements and those without (Table 3). Only one patient with MLL rearrangement showed B cell associated antigen CD19 expression and another one, T cell associated antigen CD2 expression (both were children); none revealed positive staining for CD10 or CD7 on leukemic cells (Table 3). Though patients with MLL rearrangements had a lower incidence of lymphoid-associated

MLL germline 97 36 3 18 76 35 24 25 3.5 34 8.1 22 800 38 1227 54 10 9

Total

113 35 6 21 86 36 25 25 2.9 38 8 22 500 40 1178 54 8 9

P value

0.239 0.035 0.861 0.539 1.000 1.000 0.03 0.091 0.748 0.387 0.303 1.000 0.9531 0.9007

antigen expression than others, the difference was not statistically significant. Rearrangements of Ig heavy-chain and TCR ␤-chain genes were analyzed on 54 patients. A total of three patients showed rearrangements of Ig heavy-chain gene in the leukemic cells and one of them also had TCR ␤-chain gene rearrangement; no difference existed between AML with MLL rearrangements and those without (Table 3).

Correlation of MLL rearrangements with outcome Seventy-six patients, including eight with MLL rearrangements and 68 without, received conventional combination chemotherapy for AML which contained cytosine arabinoside and one of the anthracyclines with or without other cytotoxic drugs. The complete remission (CR) rate was similar between patients with and without MLL rearrangements (50% vs 54%, Table 1). The median CR duration in patients with MLL rearrangements was 6 months (95% confidence interval, CI: 2–10 months), compared with that of 10 months (95% CI: 5– 15 months) in patients without the rearrangements (P = 0.9531). There was also no significant difference in survival time between the two groups of patients (median: 7 months, 95% CI: 0–17 months for the former group vs median: 9 months, 95% CI: 5–13 months for the latter group, P = 0.9007, Figure 2). The hazard ratio of CR duration in patients with MLL rearrangements to those without was 1.0352 (95% CI: 0.3117 to 3.4378, P = 0.9550) and that of survival time was 0.9490 (95% CI: 0.4075 to 2.2105, P = 0.9035).

Discussion MLL gene rearrangements were detected in 14% of the newly diagnosed primary AML in this study, most frequently in FAB M4 (19%) and M5 subtypes (33%) and in infant leukemia (50%). In contrast to the finding of Cimino et al17 that only two (4%) of the 52 non M4–M5 AML had MLL rearrangements, we found six (9%) of the 70 non-M4–M5 leukemia, including M1, M2 and M7, but not M3, had the abnormality. Most studies Leukemia


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Table 3

Comparison of immunophenotype and genotype between AML with MLL rearrangements and those without

Marker/Gene

HLA-DR CD34 CD13 CD33 CD11b CD14 CD15 CD19 CD10 CD2 CD7 JH T␤ a

MLL(+) patients No. studied

No. positive (%)

No. studied

15 8 15 15 15 15 8 15 15 15 14 8 8

15 (100) 2 (25) 9 (60) 12 (80) 8 (53) 8 (53) 7 (88) 1 (7)a 0 (0) 1 (7)a 0 (0) 0 (0) 0 (0)

95 33 93 94 92 93 39 94 94 95 95 46 46

P value

No. positive (%) 79 14 63 73 29 30 27 3 3 2 14 3 1

(83) (42) (68) (78) (32) (32) (70) (3) (3) (2) (15) (7) (2)

0.121 0.448 0.555 1.0 0.1 0.113 0.413 0.452 1.0 0.359 0.208 1.0 1.0

Both were children.

Figure 2 Kaplan–Meier survival curve of patients with MLL rearrangements (solid line) and those without (dashed line).

of MLL rearrangements in the literature were concentrated on infants, patients with M4–M5 AML or those with cytogenetic abnormalities involving 11q23.12,15,19,30 The incidence of MLL rearrangements in M4–M5 AML were around 10% to 30%,15,17 but its frequency in non-M4–M5 AML is unclear. Poirel et al31 observed that six (20%) of the 29 AML M1 had MLL rearrangements, but did not mention its incidence in M2, M3, M6 and M7 subtypes. The findings from their report and this study suggest that MLL rearrangements may occur in a substantial number of non-M4–M5 AML, especially in M1 subtype. M3 AML may rarely have the gene abnormality, at least at diagnosis. Because the number of patients analyzed was limited in this study, whether the high incidence of MLL rearrangements in M7 AML (33%) is a reality remains to be clarified by further studies. Recently, DNA cleavage at a specific site within the 8.3 kb MLL breakpoint cluster region during cell apoptosis has been noted by Aplan et al27,28 which results in pseudo-rearrangement of MLL gene in Southern blot analysis.29 MLL cleavage could be induced through ex vivo incubation of bone marrow cells for 24 to 60 h at room temperature.29 In addition, approximately 5% of de novo AML were shown to have MLL cleavage,32 in which the intensity of the rearranged bands corresponded to about 5% to 10% of that of the germline bands Leukemia

MLL(−) patients

in Southern blot analysis. This condition should be excluded cautiously in interpretation of MLL rearrangements. In our laboratory, mononuclear cells isolated from bone marrow aspirates were always cryopreserved on the same day of sample collection that may decrease the chance of cell apoptosis. However, DNA from three patients in this study showed pseudo-rearranged bands of MLL gene, with similar size and intensity as those reported,29,32 in Southern blot analysis (data not shown). These three cases were put into the group of patients without MLL rearrangement in analysis. Nine of the 16 patients with MLL rearrangements in this study did not have cytogenetic abnormalities at 11q23 where the MLL gene was located. This finding has been reported in 50% to 64% of AML with MLL rearrangements.20,31 Recently, partial tandem duplication of MLL was demonstrated in AML patients with normal karyotype or trisomy 11.33–35 It was also noted in some AML with chromosomal abnormalities other than 11q23.31 Caligiuri et al36 reported that 11% of AML with normal karyotype had rearrangements of MLL at diagnosis. All such cases examined showed partial tandem duplication of MLL. The incidence of MLL rearrangements in AML patients who have cytogenetic aberrations but lacking 11q23 abnormalities is not clear. In this study, 6% of the patients with normal karyotype revealed MLL rearrangements, as did 15% of those with nonspecific chromosomal abnormalities. None of the patients with specific structural chromosomal abnormalities including t(15;17), inv(16) and t(9;22) showed MLL rearrangements, but one patient with t(8;21) had this change. This was a 50-year-old man with initial white blood count of 12 800/␮l, hemoglobin level of 2.6 gm/dl and platelet count of 4000/␮l. He obtained a complete remission after chemotherapy with idarubicin and cytosine arabinoside, but died of sepsis during consolidation chemotherapy. Another patient with t(8;21) showed germline configuration of MLL gene at diagnosis, but disclosed rearranged bands by Southern blot analysis at relapse (Figure 1c). MLL rearrangements might be the secondary changes in these two AML with t(8;21). Further studies will be conducted to investigate the type of MLL gene rearrangements, including partial tandem duplication, in AML with chromosomal changes other than 11q23 abnormalities. AML patients with some specific chromosomal abnormalities show distinct immunophenotypes: an association was noted between t(15;17) and negativity to HLA-DR and CD34, t(8;21) and B cell-associated marker CD19, and t(9;22) and


lymphoid markers.23,37,38 There are only a few reports concerning immunophenotypes of leukemic cells from AML with MLL rearrangements in the literature.17,20 In contrast to ALL with MLL rearrangements which are characterized by coexpression of myeloid- and lymphoid-associated antigens on leukemic blasts,8,11 AML with MLL rearrangements in this study showed low incidence of coexpression of lymphoidassociated antigens, including CD10, CD19, CD2 and CD7, on leukemic cells (Table 3). None of the patients studied revealed rearrangements of Ig gene or TCR gene. AML patients with balanced 11q23 translocation/MLL rearrangement reported by Archimbaud et al20 also had similar low incidence of CD19 and CD10 expression, 6% and 0, respectively, as control population with normal karyotype. Expression of CD2 and CD7 was not mentioned in their report. On the contrary, coexpression of lymphoid and myeloid markers was detected in six (35%) of 17 MLL-rearranged M4–M5 cases reported by Cimino et al.17 In that report, all the lymphoid antigen-positive patients were children less than 15 years; none of the adults showed this finding. In this study, the only two MLLrearranged patients with coexpression of lymphoid antigens were also children. All the patients reported by Archimbaud et al20 were adults. It is suggested that adult AML with MLL rearrangements do not tend to coexpress lymphoid- and myeloid-associated antigens on leukemic blasts as do MLLrearranged ALL. Pediatric ALL patients with MLL rearrangements were found to have poor outcome. Little is known about the prognostic significance of MLL rearrangements in AML patients. In this study, primary AML patients with MLL rearrangement showed no differences in CR rate, CR duration and survival from those without the gene rearrangements. Bower et al15 reported that in adults with de novo AML-M4 and M5, MLL rearrangements did not identify a subgroup of patients with poorer outcome than others. On the other hand, Archimbaud et al20 demonstrated that AML patients with balanced 11q23 chromosomal anomaly and/or MLL gene rearrangements had a much poorer prognosis than patients with normal karyotype. Patients with secondary AML, in addition to primary AML, were included in their study and some cases in the analysis of outcome had only cytogenetic study but not MLL gene analysis. Further systematic studies on more patients are needed to clarify more clearly the prognostic implication of MLL rearrangements in de novo AML. It is also suggested that patients with different types of MLL rearrangements, such as translocation or partial tandem duplication, be analyzed separately. The latter has been found to be associated with poor outcome.36

Acknowledgements This study was supported in part by grants from the National Science Council of the Republic of China, NSC 87-2314B002-074 and the Children Cancer Foundation of the Republic of China, 85-11 and 86-15.

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